Bottom Line:
However, the source of the initial polarizing IL-4 remains unclear.Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells.These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.

Affiliation: Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

ABSTRACTInterleukin (IL)-4 is the most potent factor that causes naive CD4+ T cells to differentiate to the T helper cell (Th) 2 phenotype, while IL-12 and interferon gamma trigger the differentiation of Th1 cells. However, the source of the initial polarizing IL-4 remains unclear. Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells. These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.

Figure 1: IL-6 directs differentiation of CD4+ T cells into Th2 cells. (A) Total CD4+ T cells (106/ml) were purified from normal B10.BR mice and stimulated (in the presence of 5 × 105 cell/ml syngeneic APCs) with Con A (2.5 μg/ml) alone, Con A plus IL-4 (103 U/ml) or Con A plus IL-12 (3.5 ng/ml), in the absence (−) or presence of IL-6 (100 ng/ml) or IL-2 (50 U/ml). After 4 d, cells were exhaustively washed and restimulated (106 cells/ml) with Con A alone (2.5 μg/ml) in the absence of APCs or additional cytokines. Supernatants were harvested 24 h later and analyzed for IL-4 and IFN-γ production by ELISA. (B) Total CD4+ population was isolated from Cyt c TCR transgenic mice, and the naive CD4+ CD45RBhighCD44low subpopulation was purified by cell sorting. Naive CD4+ T cells were then stimulated with Con A alone, Con A plus IL-4, or Con A plus IL-12, and APCs, in the presence or absence of IL-6. After 4 d, cells were exhaustively washed and restimulated (106 cell/ ml) with Con A alone for 24 h before harvesting the supernatants for cytokine measurement. (C) Naive CD4+CD45RBhighCD44low CD4+ T cells isolated from Cyt c TCR transgenic mice were stimulated with Cyt c peptide and APCs, in the presence of medium, IL-6 (100 ng/ml), or IL-4, (103 U/ml). After 4 d, cells were exhaustively washed and restimulated (106 cell/ml) with Cyt c peptide and APCs for 24 h. (D) Total CD4+ T cells from normal B10.BR mice were stimulated with immobilized anti– CD3 mAb (5 μg/ml) and soluble anti–CD28 (1 μg/ml) in the presence of medium (−), IL-6 (100 ng/ml), or IL-4 (103 U/ml) and, after 4 d, they were restimulated with immobilized anti–CD3 mAb. (E) Total CD4+ T cells from normal B10.BR mice were stimulated with Con A and APCs in the presence of medium (−), IL-6 (100 ng/ml) (IL-6), or IL-6 (100 ng/ml) plus anti–IL-4 mAb (10 μg/ml) (IL-6 + anti–IL-4), for 4 d. Cells were then restimulated (106 cells/ml) with Con A alone as described in A.

Mentions:
IL-6 is produced by a wide spectrum of cells including fibroblasts, endothelial cells, neuronal cells, macrophages, mast cells, tumor cell lines, and CD4+ Th2 cells, but from an immunological point of view, APCs represent the major source of IL-6 (14, 19). To determine the potential role of IL-6 in differentiation of naive CD4+ T cells into effector Th1 and Th2 cells, we first analyzed the effect of exogenous IL-6. Total mouse CD4+ T cells were isolated (16) and stimulated to differentiate with Con A with or without IL-4 (Th2) or IL-12 (Th1) in the presence or absence of exogenous IL-6. After 4 d, the effector Th1 or Th2 cells were exhaustively washed, counted, and equal number of cells were restimulated with Con A for 24 h before harvesting the supernatant which was then analyzed for cytokine production. Interestingly, even in the absence of any polarizing cytokine, IL-6 directed the differentiation of the CD4+ cells to a Th2 phenotype, since the cells produce high amounts of IL-4, but not IFN-γ (Fig. 1 A). IL-6 did not modify the differentiation of the Th2 cells directed by IL-4. However, differentiation into Th1 cells by IL-12, was impaired in the presence of IL-6. Thus, Th1 cells differentiated with Con A and IL-12 in the presence of IL-6, produced less IFN-γ, and more IL-4 (Fig. 1 A). IL-6, like IL-2, has been described to be a growth factor for a number of cells (14, 15). However, only IL-6, but not IL-2, was able to modify the polarization of the CD4+ cells to Th2 phenotype (Fig. 1 A), indicating that IL-6 is involved in differentiation rather than growth of T cells. To eliminate the possibility that IL-6 could favor the expansion or IL-4 secretion of the CD4+ memory subpopulation, which has been described to display a Th2 phenotype (20), we analyzed the role of IL-6 in the differentiation of purified naive CD4+ T cells. Thus, we used CD4+ T cells from TCR transgenic mice (21), which express the α and β chain of the TCR that recognizes a pigeon Cyt c peptide; before further purification, 90–95% of the CD4+ T cells express the naive phenotype. Total CD4+ T cells were stained with anti-CD44 and anti-CD45RB mAbs and the naive CD4+ CD44lowCD45RBhigh population was isolated by cell sorting and activated with the same polyclonal stimulus, Con A, in the presence or absence of different cytokines. We observed that IL-6, in the absence of any other cytokine, was able to promote the differentiation of naive CD4+ cells to IL-4–producing cells (Fig. 1 B). In addition, we also analyzed the effect of IL-6 in the differentiation of naive CD4+ T cells stimulated by specific antigen. The presence of IL-6 during the activation with Cyt c peptide drove differentiation of naive CD4+ T cells into IL-4–producing effector Th2 cells as well or better than IL-4 (Fig. 1 C). Therefore, the modulatory effect of IL-6 in T cell differentiation is not a consequence of an expansion of the memory subpopulation.

Figure 1: IL-6 directs differentiation of CD4+ T cells into Th2 cells. (A) Total CD4+ T cells (106/ml) were purified from normal B10.BR mice and stimulated (in the presence of 5 × 105 cell/ml syngeneic APCs) with Con A (2.5 μg/ml) alone, Con A plus IL-4 (103 U/ml) or Con A plus IL-12 (3.5 ng/ml), in the absence (−) or presence of IL-6 (100 ng/ml) or IL-2 (50 U/ml). After 4 d, cells were exhaustively washed and restimulated (106 cells/ml) with Con A alone (2.5 μg/ml) in the absence of APCs or additional cytokines. Supernatants were harvested 24 h later and analyzed for IL-4 and IFN-γ production by ELISA. (B) Total CD4+ population was isolated from Cyt c TCR transgenic mice, and the naive CD4+ CD45RBhighCD44low subpopulation was purified by cell sorting. Naive CD4+ T cells were then stimulated with Con A alone, Con A plus IL-4, or Con A plus IL-12, and APCs, in the presence or absence of IL-6. After 4 d, cells were exhaustively washed and restimulated (106 cell/ ml) with Con A alone for 24 h before harvesting the supernatants for cytokine measurement. (C) Naive CD4+CD45RBhighCD44low CD4+ T cells isolated from Cyt c TCR transgenic mice were stimulated with Cyt c peptide and APCs, in the presence of medium, IL-6 (100 ng/ml), or IL-4, (103 U/ml). After 4 d, cells were exhaustively washed and restimulated (106 cell/ml) with Cyt c peptide and APCs for 24 h. (D) Total CD4+ T cells from normal B10.BR mice were stimulated with immobilized anti– CD3 mAb (5 μg/ml) and soluble anti–CD28 (1 μg/ml) in the presence of medium (−), IL-6 (100 ng/ml), or IL-4 (103 U/ml) and, after 4 d, they were restimulated with immobilized anti–CD3 mAb. (E) Total CD4+ T cells from normal B10.BR mice were stimulated with Con A and APCs in the presence of medium (−), IL-6 (100 ng/ml) (IL-6), or IL-6 (100 ng/ml) plus anti–IL-4 mAb (10 μg/ml) (IL-6 + anti–IL-4), for 4 d. Cells were then restimulated (106 cells/ml) with Con A alone as described in A.

Mentions:
IL-6 is produced by a wide spectrum of cells including fibroblasts, endothelial cells, neuronal cells, macrophages, mast cells, tumor cell lines, and CD4+ Th2 cells, but from an immunological point of view, APCs represent the major source of IL-6 (14, 19). To determine the potential role of IL-6 in differentiation of naive CD4+ T cells into effector Th1 and Th2 cells, we first analyzed the effect of exogenous IL-6. Total mouse CD4+ T cells were isolated (16) and stimulated to differentiate with Con A with or without IL-4 (Th2) or IL-12 (Th1) in the presence or absence of exogenous IL-6. After 4 d, the effector Th1 or Th2 cells were exhaustively washed, counted, and equal number of cells were restimulated with Con A for 24 h before harvesting the supernatant which was then analyzed for cytokine production. Interestingly, even in the absence of any polarizing cytokine, IL-6 directed the differentiation of the CD4+ cells to a Th2 phenotype, since the cells produce high amounts of IL-4, but not IFN-γ (Fig. 1 A). IL-6 did not modify the differentiation of the Th2 cells directed by IL-4. However, differentiation into Th1 cells by IL-12, was impaired in the presence of IL-6. Thus, Th1 cells differentiated with Con A and IL-12 in the presence of IL-6, produced less IFN-γ, and more IL-4 (Fig. 1 A). IL-6, like IL-2, has been described to be a growth factor for a number of cells (14, 15). However, only IL-6, but not IL-2, was able to modify the polarization of the CD4+ cells to Th2 phenotype (Fig. 1 A), indicating that IL-6 is involved in differentiation rather than growth of T cells. To eliminate the possibility that IL-6 could favor the expansion or IL-4 secretion of the CD4+ memory subpopulation, which has been described to display a Th2 phenotype (20), we analyzed the role of IL-6 in the differentiation of purified naive CD4+ T cells. Thus, we used CD4+ T cells from TCR transgenic mice (21), which express the α and β chain of the TCR that recognizes a pigeon Cyt c peptide; before further purification, 90–95% of the CD4+ T cells express the naive phenotype. Total CD4+ T cells were stained with anti-CD44 and anti-CD45RB mAbs and the naive CD4+ CD44lowCD45RBhigh population was isolated by cell sorting and activated with the same polyclonal stimulus, Con A, in the presence or absence of different cytokines. We observed that IL-6, in the absence of any other cytokine, was able to promote the differentiation of naive CD4+ cells to IL-4–producing cells (Fig. 1 B). In addition, we also analyzed the effect of IL-6 in the differentiation of naive CD4+ T cells stimulated by specific antigen. The presence of IL-6 during the activation with Cyt c peptide drove differentiation of naive CD4+ T cells into IL-4–producing effector Th2 cells as well or better than IL-4 (Fig. 1 C). Therefore, the modulatory effect of IL-6 in T cell differentiation is not a consequence of an expansion of the memory subpopulation.

Bottom Line:
However, the source of the initial polarizing IL-4 remains unclear.Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells.These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.

Affiliation:
Section of Immunobiology, Yale University School of Medicine, New Haven, Connecticut 06520-8011, USA.

ABSTRACTInterleukin (IL)-4 is the most potent factor that causes naive CD4+ T cells to differentiate to the T helper cell (Th) 2 phenotype, while IL-12 and interferon gamma trigger the differentiation of Th1 cells. However, the source of the initial polarizing IL-4 remains unclear. Here, we show that IL-6, probably secreted by antigen-presenting cells, is able to polarize naive CD4+ T cells to effector Th2 cells by inducing the initial production of IL-4 in CD4+ T cells. These results show that the nature of the cytokine (IL-12 or IL-6), which is produced by antigen-presenting cells in response to a particular pathogen, is a key factor in determining the nature of the immune response.